Contents

Examine candidate clones

Introduction

Yesterday a few milliliters of LB+AMP broth were inoculated with some candidate colonies and the tubes were grown overnight at 37°C. Ampicillin was included in the broth to ensure that the cells would maintain the plasmid DNA. Today the cells will have grown to high density and the plasmid DNA will have undergone many replications. All the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another), since the culture began with a single colony and that colony grew from a single transformed cell. Next, you must isolate the plasmid DNA from the candidates and determine if any of the plasmids contain the truncated EGFP gene.

To isolate the plasmid from the overnight cultures, you will perform what is commonly called a “mini-prep.” This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA and cellular debris, allowing the plasmid DNA to be studied further.

In the traditional mini-prep protocol, which you will perform today, the media is removed from the cells by centrifugation. The cells are resuspended in “Solution I” which contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and SDS is then added. The base denatures the cell’s DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by the potassium acetate in “Solution III.” At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution, effectively separating plasmid DNA from the chromosomal DNA and the proteins and lipids of the cell.

Protocol

Two notes:

The tissue culture facility in the lab can accommodate no more than 12 students at a time. Half the class will begin by performing the tissue culture portion of today’s experiment (part 3) while the other half will begin in the main lab preparing their samples to load on an agarose gel (parts 1 and 2).

Sometime before you leave today you should count or estimate the number of colonies that arose from your transformation reactions.

Part 1: Plasmid miniprep

One of the teaching faculty has set up four overnight LB + AMP cultures of bacteria for you to work with. Three of these cultures have colonies from the ligation reactions you have been preparing (if your reactions were unsuccessful then you have been provided with candidates from other reactions). One of these cultures has grown from a colony of bacteria with pCX-NNX. Plasmid from this culture will serve as a control for today’s digests and will provide a suitable comparison pattern on the agarose gel you will run next week.

Label your three candidate cultures 1, 2 or 3 and four eppendorf tubes (control, 1, 2, or 3). Vortex the bacteria then pour some into the appropriate eppendorf tube so that the tubes are almost full. If you are nervous about pouring the liquid, you can use your P1000 to pipet 750 μL into each eppendorf twice. Either way, the eppendorf should be quite full when you try to close the cap. You can wear gloves to keep the bacteria from splashing your skin or you can wash your hands after closing all the caps.

Balance the tubes in the microfuge, and then spin them for one minute.

Aspirate the supernatant, as shown, removing as few cells as possible.

Aspirate the supernatant, as shown, removing as few cells as possible

Resuspend the cells in 100 μl of Solution I, changing tips between samples.

Prepare Solution II by mixing 500 μl of 2% SDS with 500 μl of 0.4M NaOH in an eppendorf tube. Add 200 μl of Solution II to each sample and invert the tubes five or six times to mix. In some cases the samples may appear to "clear" but don't worry if you don't see a big change. Place the tubes on ice for at least 2 minutes.

Add 150 μl of Solution III to each tube and immediately vortex each tube for 10 seconds with your vortex set at the highest setting. White clumps should appear in the solution after you vortex it. Place the tubes in the room temperature microfuge and spin them for 4 minutes.

While the tubes are spinning, label another set of eppendorf tubes with the plasmid names and your team color.

A white pellet should be visible when you remove your tubes from the microfuge. Use your P1000 to transfer 400 μL of each supernatant to the appropriate clean eppendorf tube. It's OK to leave some of the supernatant behind. Avoid transferring any of the white pellet.

Add 1000 μl of room temperature 100% ethanol to each new tube. The tubes will be quite full. Close the caps and invert the tubes at least five times to thoroughly mix the contents.

Microfuge the samples for 2 minutes. It is important to orient your tubes in the microfuge this time since the pellets from this spin will be barely visible.

Remove the supernatants using your P1000 or the aspirator, but be careful not to disturb the pellet of plasmid DNA that is at the bottom of the tube. Remove as much of the supernatant as possible but you do not need to remove every drop since you will be washing the pellet in the next step.

Add 500 μl of 70% ethanol to each pellet. Spin the samples one minute, orienting the tubes in the microfuge so you will know where to find the pellet. Immediately remove the supernatant with your P1000, making sure to keep the tip on the side of the tube that doesn't have your pellet. Remove as much liquid as possible, using your P200 set to 100 μL, to remove the last few droplets.

To completely dry the pellets, place your rack in the hood with the caps open for a few minutes. When the pellets are completely dry, add 50 μl of sterile water to each sample and vortex each tube for 2 X 30 seconds to completely dissolve the pellets. The liquid can be brought back to the bottom of the tubes by spinning them in the microfuge for a few seconds.

Part 2: Diagnostic digests

You will perform a diagnostic digest on three of the four plasmids you have prepared (one sample will be the pCX-NNX parent and you'll choose 2 of your 3 candidates to pursue) to see if any have the PCR insert. Use information from the lab manual, the NEB catalog and the plasmid maps you’ve drawn to choose the enzymes you’ll use. The following table may be helpful as you plan your work.

Diagnostic Digest

Enzyme 1 only

Enzyme 2 only

uncut

Plasmid DNA

5 μl

5 μl

5 μl

5 μl

10X NEB buffer

2.5 μl of buffer#_____

2.5 μl of buffer#_____

2.5 μl of buffer#_____

2.5 μl of buffer#_____

Enzyme

0.25 μl of _____

0.25 μl of _____

2nd Enzyme

0.25 μl of _____

0.25 μl of _____

H2O

For a total volume of 25 μl

Prepare a reaction cocktail for each of the above reactions (uncut, singly cut with enzyme 1, singly cut with enzyme 2 and doubly cut with Enzyme 1 and Enzyme 2) that includes water, buffer and enzyme. Prepare enough of each cocktail for 4 digests. Leave the cocktails on ice.

Aliquot 5 μl of plasmids (pCX-NNX and 2 of your three candidates) into a sets of well-labeled eppendorf tubes. The labels should include the plasmid name, the enzymes to be added and your team color.

Add 20 μl of the appropriate cocktail to each tube. Flick the tubes to mix the contents, then incubate the mixtures at 37°C.

Before leaving lab today, please add 2 μl of loading dye to each of the digests you have assembled. Store the digests and the DNA at –20°C and return the remainder of your bacterial cultures to one of the teaching faculty.

Part 3: Tissue Culture

Each of you will have a 60 mm dish of mouse embryonic stem (MES) cells that you will use to seed a six-well dish. You and your partner will seed the dishes at different concentrations so you should decide who will seed at 1:100 and who will seed at 1:400.

Prewarm all the required reagents in the water bath.

Look at your cells as you remove them from the incubator. Look first at the color and clarity of the media. Fresh media is reddish-orange in color and if the media on your cells is yellow or cloudy, it could mean that the cells are overgrown, contaminated or starved for CO2. Next look at the cells on the inverted microscope. Note their shape and arrangement in the dish and how densely the cells cover the surface.

Move the cells into the sterile hood, as well as the PBS, trypsin, and media that you will need. One of the greatest sources for TC contamination is moving materials in and out of the hood since this disturbs the air flow that maintains the sterile environment inside the hood. Anticipate what you will need during your experiment to avoid moving your arms in and out of the hood while your cells are inside.

Aspirate the media from the cells.

Wash the cells by adding 3 ml PBS with a 5 ml pipet. Tip the dish in all directions to rinse all the cells, and then aspirate the liquid out of the dish.

Repeat the PBS wash, leaving the cells dry for the next step.

To dislodge the cells from the dish, you will add trypsin, a proteolytic enzyme. Using a 2 ml pipet, add 0.5 ml of trypsin to the dish. For one minute precisely (use your timer), tip the dish in each direction to distribute the trypsin over the cells then aspirate the trypsin off the cells. Incubate the cells (“dry”) at 37° for 10 minutes, again using your timer to precisely time this incubation.

While you are waiting, you can add 1 ml of gelatin to each well of a six-well dish. This should be done in the sterile hood with sterile technique. The gelatin will be removed before you seed the dish with your MES cells but it is important to pre-treat the dish this way. The gelatin must remain in the wells for at least 10 minutes.

With a 5 ml pipet, add 4 ml of media to the trypsinized MES cells and pipet the liquid up and down (“triterate”) to remove the cells from the plastic and suspend them in the liquid. Remove a small amount of the liquid to an eppendorf tube and take it to the inverted microscopes.

Fill one chamber of a hemocytometer with 10 ul of the cell suspension. This slide has an etched grid of nine large squares. The square in the center is further etched into 25 squares each with a volume of 0.1 ul and 16 tiny chambers (4x4 pattern). The concentration of cells in a sample can be determined by counting the cells that fall within the 4x4 pattern and then multiplying by 10,000 to determine the number of cells/ml. You should count the cells in the four corner squares of the 25 square grid, then average the numbers to determine the concentration of cells in your suspension.

Counting cells using a hemocytometer

You and your partner will seed at different concentrations. Decide if you will try the 1:100 or 1:400 dilution and add the appropriate amount of cell suspension to 10 ml of media in a 15 ml conical tube.

Remove the gelatin from the six-well dish you have prepared and add 3 ml of your cell dilution to each well. Be sure to label your dish with your name, today’s date, the cell line (called “J1”) and the type of media you have used. Return your cells to the incubator.

Aspirate any remaining cell suspensions to destroy them and clean up the hood. Dispose any vessels that held cells in the Biohazard waste and any sharps in the grey bins. The next group who uses your hood should find the surfaces wiped down, no equipment left inside, the sash closed and the germicidal lamp on.

If you are done early, try to calculate the number of cells you'd expect to see next time in each well of your six well dish. Base your calculation on the following three observations:

only 25% of the cells are able to stick and proliferate (this is called a 25% plating efficiency)

AND

the doubling time for the cells is 24 hours,

AND

the cells take 24 hours to recover from trypsin treatment before they begin doubling.